The affinity chromatography method denoted IMAC (Immobilized Metal Ion Affinity Chromatography), pioneered by Porath and coworkers (Nature 258:598, 1975), has been in use for protein purification for decades. The principle of IMAC lies in the fact that many metal ions can form coordination bonds between oxygen and nitrogen atoms of amino acid side chains in general and of histidine, cysteine, and tryptophan in particular. The use of IMAC started to increase very considerably in the late 1980:ies with the introduction of methods to genetically modify proteins of interest, so that they contained more histidine residues than coded for by the natural gene, for increased affinity for immobilized metal ions. Such “histidine-tags” can have consecutive His residues, usually 6-10×His, or be various short peptide segments containing His residues together with other amino acid residues (Hochuli et al., Bio/Technology 6:1321, 1988; Chaga et al., J Chromatogr A 864:247, 1999). A histidine-tag is accordingly used with the aim of giving the recombinant target protein an affinity for immobilized metal ions that is higher or considerably higher than that of any natural protein that can be present together with the non-purified target protein, e.g. in a cell lysate.
Notably, the histidine-tags of such recombinant proteins are not only being used for affinity purification purposes, but also for immobilization of the proteins via immobilized chelators and their metal ions for protein-detection purposes (see Discussion below). It should be mentioned that in all these applications, the use of imidazole as a competitive agent that, at an optimized concentration, decreases unwanted interactions between non-tagged components and metal-ion charged chelators, can be advantageous. This is well known and much used.
Various metal ions have been in use for the purposes here mentioned, notably ions of Ni, Cu, Co, Zn, Ca, Mg, Fe, Ga, Sc and, in medical diagnostics applications with chelator conjugates, radioisotopes of Y, In, Tc, Cu, Ga and others.
Chelators vary in structure and properties, notably in how many of the coordination positions of a metal ion the chelator occupies, or is considered to occupy. Accordingly, chelators can be denoted tri-dentate, tetra-dentate, or pentadentate. This coordination position property of chelators is obviously of importance for the strength of binding of metal ions. Much used and commercialised chelators are iminodiacetic acid (IDA) which is a tri-dentate chelator, and nitrilotriacetic acid (NTA) which is a tetra-dentate chelator. Pentadentate chelators have been less used, but have been described in the literature, e.g. TED, tris(carboxymethyl)ethylenediamine (Porath & Olin, Biochemistry 22:1621, 1983) and IMAC resins with pentadentate chelators are also commercially available.
It should be noted that careful chelator synthesis and, e.g., coupling to a matrix cannot be considered trivial, in case a single homogeneous chelating ligand is desired. Adsorbents/chelators that exist/are prepared as single species, and are not mixtures, are obviously well adapted to show as distinct properties as is possible, to the benefit of the intended use. The non-triviality of preparation of IMAC adsorbents can be especially evident in cases where a ligand is synthesized step-wise in situ, i.e. on a matrix (McCurley & Seitz, Talanta 36:341, 1989); see also below. In other cases where a chelator is synthesized in solution and then purified to remove unwanted side products, also the subsequent coupling procedure may need to be performed with skill and experience, to give only a single mode of chelator immobilization.
There are several potential advantages that could be attributed to pentadentate chelators. All protein binding to the complexed metal ion should be weakened compared to tri- and tetra-dentate chelator since the number of metal ion coordination sites available for a biomolecule is lower. Thus, many non-tagged proteins that show some affinity for metal ion complexes with tri- and tetradentate chelators may not bind at all or only weakly, leading to higher selectivity for histidine-tagged proteins. This could be of particular importance for low-level target protein expression, where competitive displacement of weak, unwanted binders by an excess of the strongest binder, namely the histidine-tagged protein, is difficult to use to an advantage at IMAC purification.
Furthermore, the stronger binding of metal ions to a pentadentate chelator will decrease the loss of the ions during chromatography, decrease the risk for contamination of the purified protein with traces of metal ions (which may be harmful to the protein), and make the chromatography resin re-usable without the need for re-charging with new metal ions before the next use. Such aspects are especially important for feeds (=samples applied to the chromatographic column) like animal cell culture media and buffers that are “aggressive”, i.e., that tend to remove the immobilized metal ions. Also when substances that disturb the purification by interacting with the metal ions are present in feeds and/or buffers, e.g. some disulfide-reducing agents, it should be an advantage to use IMAC resins that have a pentadentate chelator. Less contamination of work environments and wastewater with released metal ions would also be a consequence of a firm metal ion binding.
Prior Art Related to Monomeric Pentadentate Chelators
Haner et al. (Analytical Biochemistry 138:229, 1984) describe pentadentate chelator adsorbents produced by linking EDTA covalently to amino-agarose. A cobalt complex of EDTA was used for coupling to the polymer resin. The described use of the resins was the removal of unwanted Ca′ ions.
Similarly to Haner et al., pentadentate chelators on amine-derivatized non-toxic polymers have been described by Schellenberg et al., Eastern Virginia Medical School, Norfolk, US (U.S. Pat. No. 6,020,373) and by Li et al., Changchun Institute of Applied Chemistry, China (CN 1,966,088). These inventions and the uses thereof were in the fields of medicine and medical diagnostics.
EP 2 022 561 (Gorlich & Frey, Max-Planck-Gesellshaft, Munchen, DE) describes a method for binding of polycarboxylic acids such as EDTA or EGTA, to a solid phase comprising amino groups. It was claimed that single-point binding, i.e., via only one carboxyl group predominated under certain conditions.
Also WO 2009/008802 (Andersson et al., GE Healthcare, Uppsala, SE) relates to easy and rapid coupling of precursors of pentadentate chelators to amine-containing polymers, to give resins with very strong binding of metal ions, which is advantageous for many instances of biomolecule adsorption and/or detection.
Prior Art Related to Di- or Trimeric Chelators for Binding of Biomolecules
Thus, all the above publications relate to monomeric pentadentate chelators. There are also several published descriptions of the preparation and use of di- and trimeric chelators holding two or three adjacent metal ions, in the field of immobilization of (histidine-tagged) proteins for interaction studies by surface plasmon resonance analysis or similar analysis, and mostly or exclusively describing NTA-based chelators, i.e. tetradentate chelators. Notable in this field are several publications by Piehler and coworkers, Johann Wolfgang Goethe University, Frankfurt, DE (e.g., Analytical Chemistry 77:1092, 2005; US 2008/0038750) and by Ebright & Ebright et al., Rutgers University, New Brunswick, US (J Am Chem Soc 123:12123, 2001; U.S. Pat. No. 7,371,745).
Surprisingly, we have not found any prior art describing pentadentate dimeric chelators in general, nor any descriptions of pentadentate dimeric chelators that display unusually strong binding of metal ions, nor any use of such chelators for binding to, or adsorbing biomolecules.